In this study, we have extended and completed a previous investigation (P. Messner, R. Christian, J. Kolbe, G. Schulz, and U. B. Sleytr, J. Bacteriol. 174:2236-2240, 1992) in which we demonstrated for the first time in prokaryotic organisms the presence of a novel O-glycosidic linkage via tyrosine. The surface layer glycoprotein of the eubacterium Clostridium thermohydrosulfuricum S102-70 is arranged in a hexagonal lattice, with center-to-center spacings of approximately 16.3 nm. Molecular weight determination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both glycosylated and chemically deglycosylated surface layer glycoprotein showed values for the monomeric subunits of 94,000 and 87,500, respectively. Glycopeptide fractions obtained after exhaustive pronase digestion of purified, intact glycoprotein were isolated by reversed-phase liquid chromatography. One- and two-dimensional nuclear magnetic resonance studies, together with chemical analyses and plasma desorption time-of-flight mass spectrometry, were used to elucidate the structure of the hexasaccharide moiety linked by the novel O-glycosidic linkage to tyrosine. The combined evidence suggests the following structure: beta-D-Galf-(1-->3)-alpha-D-Galp- (1-->2)-alpha-L-Rhap-(1-->3)-alpha-D-Manp-(1--3)-alpha-L- Rhap-(1-->3)-beta- D-Glcp-(1-->4)-L-Tyr
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